The middle and small tumor antigens of polyoma virus and the small tumor antigen of SV40 form complexes with protein phosphatase 2A in virally infected cells. Studies with mutants of polyoma middle T antigen have shown that this interaction, along with those of several other cellular proteins, is necessary but not sufficient for transformation. Although not capable of causing transformation by itself, SV40 small T enhances transformation by large T antigen. Current evidence suggests that all of the biological effects of SV40 small T are mediated by interaction with phosphatase 2A. We have developed a reconstituted system consisting of purified phosphatase 2A, or its subunits, and recombinant small T. Small T and phosphatase 2A form complexes in vitro, and this interaction inhibits phosphatase activity in a manner similar to that caused by the B subunit normally present in the enzyme. Our current model proposes that the biological effects of small T are due to changes in the activity of phosphatase 2A toward a selected set of substrates, without a major change in the overall level of type 2A activity. This project will study the interaction of SV40 small T with protein phosphatase 2A and the biochemical effects of formation of this complex. We will also attempt to identify potential substrates whose phosphorylation are influenced by this interaction. A series of small T mutants will be prepared that have altered biochemical and transformation helper functions. These mutants will be used in reconstitution assays to determine the molecular basis for the small T-phosphatase interaction. The effects of small T (and small T mutants with altered biological properties) on phosphatase 2A activity will be tested with a series of model substrates. We will also determine if dephosphorylation of potential in vivo substrates including SV40 large T, the retinoblastoma protein, and p53 is altered by interaction with small T. These studies should allow us to determine if the effects of small T on transformation are due to interaction with phosphatase 2A. The involvement of a cellular exchange factor in the interaction of small T with phosphatase 2A will be tested. A previously characterized biochemical activity of small T is the transactivation of certain genes. We will identify potential substrates of the small T/phosphatase 2A comp involved in trans-activation of the adenovirus E2 and T cell receptor gamma-chain genes, using in vitro transcription assays.